1. Field of the Invention
The present invention relates to intraplexed assays, and more particularly to a device and method for improving intraplexed assays to increase precision and predictability of suspended microarray assays.
2. Background
A number of patented technologies exist for use in suspended microarray assays. These include substrate microparticles of various kinds, ways to differentiate between said microparticles, methods of processing the array data, and what is called multiplexing of assays.
There is currently a method for improving assay precision and reliability called intraplexing, as described in U.S. Pat. No. 7,501,290, granted to the present applicant and incorporated herein as if set out in full.
Intraplexing improves upon of the prior art multiplexing techniques by intentionally using more than one particle set targeted at the same analyte within a sample. As disclosed in the '290 patent, intraplexing may employ multiple sets of particles which have different sensitivities to the same analyte in order to better determine analyte concentration. Intraplexing may also employ multiple identically sensitive assays to improve precision in analyte quantification. These techniques can be used to eliminate instrumental variances and make accurate estimates of the true concentration of analytes without necessarily requiring calibration standards be run with every multi-well assay that is performed.
To define intraplexing clearly, and to clarify the Applicant's improvement upon intraplexing, the following terminology is used for purposes of this patent application. A single particle is called an SMP (suspended microarray particle.) A set of microspheres that are all labeled with the same classifier is called an SMPCS (suspended microarray particle category set). An SMPCS is composed of SMPs, which are identically identifiable by use of a flow cytometer or other such devices as are known in the art.
Intraplexing also employs what is called a superset, or SMPCS-IDG (suspended microarray particle category set identical group). In the intraplexing methods disclosed in the '290 patent, an SMPCS-IDG comprises a set of different SMPCSs which are all coated with the same reagent(s) so as to make them identical in sensitivity to the analyte being assayed. In the context of the current invention, an SMPCS-IDG comprises a set of different SMPCSs, which are all coated with the same reagent(s) so as to make them identical in affinity for the analyte being assayed. Bearing this introduction in mind, these terms are discussed in more detail below.
A suspended microarray system uses a population of suspended microarray particles (SMPs), all of which have had their surfaces coated with an assay. After an assay protocol, these SMPs are run through a flow cytometer, which has a flow cell that differentiates individual SMP events as they go by. This flow cell concurrently differentiates individual SMPs, identifies which SMPCS an SMP belongs to, and determines whether the SMP has captured any analyte. Conventionally, most of these SMP-based assays use fluorescent reporter molecules to provide signal, but there are other methods. If multiple SMPCSs are present in a well, then more than one analyte can be assayed simultaneously in the same assay plate well. This is termed multiplexing of assays, and it is a primary selling point for the current generation of suspended microarray systems, such as those sold by Luminex out of Austin, Tex.
Multiplexing suffers from multiple stochastic and non-stochastic sources of error as detailed in the '290 patent. As such, it generally requires replication of samples. Intraplexing as disclosed in the '290 patent overcomes these problems by employing a plurality of SMPCS readings for each assay. Intraplexing also enables statistically significant results to be attained from assays applied to single well samples by generating multiple results from a single well. Intraplexing represents a significant improvement over multiplexing, but there is room for further improvement of the intraplexing method. The current invention seeks to improve upon the previously disclosed intraplexing method by improving precision and reliability of the assay.
It is therefore a first objective of the instant application to improve precision and reliability beyond that disclosed in the '290 patent.
It is a second objective of the instant application to provide a greater degree of redundancy, and hence, of statistical significance, resolving concentration of analytes.
It is a third objective of the instant application to provide an alternative method for improving precision and reliability substantially similar to the '290 patent.